Thermal Stability of Globins: Implications of Flexibility and Heme

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Computational Biochemistry

Thermal Stability of Globins: Implications of Flexibility and Heme Coordination Studied by Molecular Dynamics Simulations Laia Julió Plana, Alejandro D Nadra, Dario A Estrin, F. Javier Luque, and Luciana Capece J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.8b00840 • Publication Date (Web): 05 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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Journal of Chemical Information and Modeling

Thermal Stability of Globins: Implications of Flexibility and Heme Coordination Studied by Molecular Dynamics Simulations

Laia Julió Plana1, Alejandro D. Nadra2, Dario A. Estrin1, F. Javier Luque3,4 and Luciana Capece1* 1Departamento

de Química Inorgánica, Analítica y Química Física, Facultad de

Ciencias Exactas y Naturales, Universidad de Buenos Aires / Instituto de Química Física de los Materiales, Medio Ambiente y Energía (INQUIMAE-CONICET), C1428EGA, Buenos Aires, Argentina. 2Departamento de Fisiología y Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires / IQUIBICEN-CONICET, C1428EGA, Buenos Aires, Argentina. 3Department of Nutrition, Food Sciences and Gastronomy, Faculty of Pharmacy and Food Sciences, University of Barcelona, Campus Torribera, 08921, Santa Coloma de Gramenet, Spain 4Institute of Biomedicine (IBUB) and Institute of Theoretical and Computational Chemistry (IQTCUB), University of Barcelona, 08028, Barcelona, Spain. *To whom correspondence should be addressed: [email protected].

Keywords: globins, molecular dynamics, thermostability, heme coordination, structural fluctuations, CD region, protein flexibility.

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Abstract Proteins are sensitive to temperature, and abrupt changes in the normal temperature conditions can have a profound impact in both structure and function, leading to protein unfolding. However, adaptation of certain organisms to extreme conditions raises questions about the structural features that permit to preserve the structure and function of proteins in these adverse conditions. To gain insight into the molecular basis of protein thermostability in the globin family, we have examined three representative examples: human neuroglobin, horse heart myoglobin and Drosophila hemoglobin, which differ in their melting temperature and the coordination state of the heme iron in absence of external ligands. In order to elucidate the possible mechanisms that govern the thermostability in these proteins, microsecond-scale classical molecular dynamics simulations were performed at different temperatures. Structural fluctuations and essential dynamics were analyzed, indicating that the flexibility of the CD region, which includes the two short C and D helixes and the connecting CD loop, is directly related with the thermostability. We observe that a larger inherent flexibility of the protein produces higher thermostability, probably concentrating the thermal fluctuations observed at high temperature in flexible regions, preventing unfolding. Globally, the results in this work improve our understanding of thermostability in the globin family.


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Introduction Globins constitute a widely distributed family of proteins responsible of a great variety of biological functions in all kingdoms of life 1–3. They contain heme as a prosthetic group, which is found inserted into the core of the protein, and generally fold in a globular 3over-3 sandwich motif formed by helices labeled A, B, E, F, G and H (Figure 1A). The remaining short helices, C and D, together with the CD loop, form the CD region.

Figure 1. A) Cartoon representation of the crystal structure of neuroglobin, taken from PDB 1OJ6, as an example of the conserved 3-over-3 globin fold. Each helix is colored differentially and depicted in the figure. B) 6c  5c equilibrium displayed by the heme group in hexacoordinated globins. XO represents a generic small ligand as O2, CO or NO.



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The two regions that are delimited by the plane of the porphyrin ring are commonly known as proximal and distal sites. The iron in the heme group is bonded by four nitrogen atoms of the porphyrin ring in its equatorial coordination sites and a conserved histidine residue in the proximal site (named HisF8 for its position in the F helix), forming the characteristic pentacoordinate state (5c). On the other hand, the distal site usually binds small external ligands, as O2, CO, NO, or H2S, which is closely related to protein function4. In some cases, a protein residue coordinates the iron reversibly (especially in the ferrous state) in the distal site, forming an hexacoordinated (6c) protein (Figure 1B). Frequently, the residue that occupies the distal site in 6c globins is a histidine residue in the 7th position of the E helix, HisE7, resulting in a bis-histidyl hexacoordinated species. However, residues located in other positions, as E11, might also coordinate the iron as observed in other globins (specially truncated hemoglobins5). This process introduces an additional mechanism to control the function of globins, since binding of exogenous ligands requires the cleavage of the Fe-distal residue bond. This finely tuned equilibrium between 6c and 5c states can be experimentally characterized by the equilibrium constant KHis=kon,His/koff,His, where kon,His and koff,His denote the kinetic constants for His binding and unbinding, respectively. In previous works6–8 we have shown that protein flexibility, especially in the CD region (Figure 1A, region in red), is a key factor in the regulation of the 6c5c equilibrium. It is well known that proteins are sensitive to environmental conditions, including changes in temperature and pressure, among others. In particular, a rise in temperature can lead to alterations in protein stability and flexibility, and even to protein unfolding. However, differential behavior towards temperature is observed, even in proteins that display high sequence identity and exhibit very similar 3-dimensional structure. This is the case, for instance, of homologous enzymes of thermally adapted and non-adapted organisms9–11. 4 ACS Paragon Plus Environment

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Interestingly, proteins that belong to thermophilic microorganisms are able to retain their structure and function even at high temperature conditions12,13. Thus, adaptation to extreme temperature is not easy to explain. From the folding landscape theory, the stability of the native state is governed by the free energy gap between the ensemble of micro configurations corresponding to folded and unfolded states. Experimentally, this is commonly related with the melting temperature of the protein14. In this context, denaturation involves a large loss of enthalpic stabilization but, on the other hand, a large gain in entropic stabilization. Thus, a larger number of contacts in the native state, which is typically associated to a larger rigidity of the protein structure, will favor the enthalpic contribution, and benefit high thermal stability15–22. On the other hand, focusing on the entropic contribution to the thermal stability, a larger conformational entropy in the folded state, which is typically associated to higher flexibility of the protein structure, will also benefit thermal stability23. Indeed, a more recent work that compares thermal stability between truncated hemoglobins from mesophilic and thermophilic organisms indicate that proteins with larger flexibility display larger thermal stability24. Thus, it is not straightforward to predict a priori the effect of protein flexibility in thermal stability. In this work we intend to gain insight into the molecular basis of protein thermostability in globins. When focusing in the globin family, the picture is even more complicated due to the presence of the heme group and the different coordination states that the iron in the heme group can adopt.

In order to perform this study, we have selected three

representative examples: human brain neuroglobin (Ngb), horse heart myoglobin (Mb) and Drosophila hemoglobin (DroHb). These examples differ in their melting temperature (Tm) and in their coordination state in absence of external ligands. Ngb has been reported to have a Tm of 373K (or 363K in the presence of a disulfide bridge in the CD region),



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being the most thermostable out of the three proteins. The second example is Mb, with a Tm of 354K, and finally DroHb has been reported to have a Tm of 349K 25. Regarding the 6c5c equilibrium, DroHb populates both coordination states with a KHis of 18, Mb displays only the 5c state, with a KHis

Thermal Stability of Globins: Implications of Flexibility and Heme

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